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Open AcceResearch articleConstitutive RB1 mutation in a child conceived by in vitro fertilization: implications for genetic counselingRaquel H Barbosa1,2, Fernando R Vargas1,3, Evandro Lucena4, Cibele R Bonvicino1 and Héctor N Seuánez*1,5Address: 1Genetics Division, Instituto Nacional de Câncer, Rua André Cavalcanti, 37, 4th floor, 20231-050 Rio de Janeiro, RJ, Brazil, 2Post-graduate Program in Oncology, Instituto Nacional de Câncer, 20231-050 Rio de Janeiro, Brazil, 3Department of Genetics, Universidade Federal do Estado do Rio de Janeiro, 22290-240 Rio de Janeiro, Brazil, 4Paediatric Service, Instituto Nacional de Câncer, 20230-030 Rio de Janeiro, Brazil and 5Department of Genetics, Universidade Federal do Rio de Janeiro, 21941-901 Rio de Janeiro, Brazil
Email: Raquel H Barbosa - [email protected]; Fernando R Vargas - [email protected]; Evandro Lucena - [email protected]; Cibele R Bonvicino - [email protected]; Héctor N Seuánez* - [email protected]
* Corresponding author
AbstractBackground: The purpose of this study was to identify mutations associated with bilateralretinoblastoma in a quadruplet conceived by in vitro fertilization, and to trace the parental originof mutations in the four quadruplets and their father.
Methods: Mutational screening was carried out by sequencing. Genotyping was carried out fordetermining quadruplet zygosity.
Results: The proband was a carrier of a novel RB1 constitutive mutation (g.2056C>G) which wasnot detected in her father or her unaffected sisters, and of two other mutations (g.39606 C>T andg.174351T>A) also present in two monozygotic sisters. The novel mutation probably occurred denovo while the others were of likely maternal origin. The novel mutation, affecting the Kozakconsensus at the 5'UTR of RB1 and g.174351T>A were likely associated to retinoblastoma in theproband.
Conclusion: Molecular diagnosis of retinoblastoma requires genotypic data of the family fordetermining hereditary transmission. In the case of children generated by IVF with oocytes from ananonymous donor which had been stored in a cell repository, this might not be successfullyaccomplished, making precise diagnosis impracticable for genetic counseling.
BackgroundRetinoblastoma (RB; MIM #180200) is the most commonintraocular pediatric tumor, with an incidence of 1/15,000–25,000 live births. It results from mutationalinactivation of both alleles of the tumor suppressor RB1gene that encodes a nuclear phosphoprotein (pRB)involved in the control cell cycle [1-3]. In the hereditary
form, accounting for 40% of RB patients, an RB1 germlinemutation is transmitted as an autosomal dominant traitwith high penetrance (90%), resulting in a 45% risk ofoccurrence in the offspring, with increased risk for second-ary malignancies. In the sporadic form, mutational inacti-vation of both RB1 alleles arises from somatic events inretinal cells, without germline alterations [4].
Published: 29 July 2009
BMC Medical Genetics 2009, 10:75 doi:10.1186/1471-2350-10-75
Received: 26 February 2009Accepted: 29 July 2009
This article is available from: http://www.biomedcentral.com/1471-2350/10/75
© 2009 Barbosa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Genetic alterations frequently resulting from RB1 inacti-vation involve chromosome rearrangements affecting the13q14 region (deletion, translocation), nucleotidechanges (substitutions, deletions, insertions and duplica-tions), exonic deletions (single or multiple); loss of heter-ozygosity [5], or CpG island hypermethylation of the RB1promoter region [6]. Epigenetic mechanisms playing arole in some RB patients include differential methylationof chromosome 13q around RB1 [7] and preferential lossof maternal alleles in sporadic cases, suggesting a latentimprinting [8].
Identification of RB1 mutations in hereditary RB providesaccurate risk prediction and valuable genetic counselingfor patients and their families. Furthermore, the nature ofa mutation can determine genetic penetrance, diseasepresentation and prognosis [9].
Recent investigations suggest an apparent associationbetween in vitro fertilization (IVF) and genetic syndromes,including retinoblastoma [8]. It has been postulated thatembryos in culture may acquire epigenetic defects as aresult of abnormal environmental conditions which maylead to aberrant phenotypes. These studies illustrated acomplex problem involving legal and ethical aspects andprivacy protection in the field of assisted reproductivetechnologies (ART), with potential implications in thegenetic counseling of retinoblastoma.
We herein describe a novel constitutive mutation at the 5'UTR of RB1 in a patient with bilateral retinoblastoma,who also carried two other constitutive mutations alsopresent in two of her sisters conceived by IVF.
MethodsPatients and samplesWe studied a family of female quadruplets (A, B, C and D)conceived by IVF of oocytes from one anonymous donorwith sperm of their father. One quadruplet presentedbilateral retinoblastoma diagnosed by current ophthal-mological and histopathological criteria at one month ofage. The proband (A) was referred to the Genetic Coun-seling Program of the Instituto Nacional de Cancer (Riode Janeiro, Brazil) and subsequently selected for muta-tional screening, together with her three sisters and theirfather. All quadruplets have been followed by periodicalophthalmological examinations to present.
Blood samples were obtained from the quadruplets andtheir father. Samples and information on the family wereobtained with an informed consent. Tumor samples werenot available for analysis. With respect to privacy and ano-nymity required for IVF, blood samples could not beobtained from the female donor. This study was approvedby local Ethics Committee and followed the tenents andguidelines of the Declaration of Helsinki.
Mutation screeningRB1 exons 2–27 were PCR-amplified in individual reac-tions containing 100 ng DNA, 0.4 pm of each primer [for-ward and reverse previously reported [5]], 0.15 mM ofeach dNTP, 3 mM MgCl2, 1× PCR buffer (Promega Corpo-ration, Madison, Wisconsin, USA) and 1 U of Taq DNApolymerase (Promega Corporation) in 50 μl reactions.PCR was performed with 40 cycles at 94°C (1 min), 54°C(40 sec) and 72°C (35 sec) in a programmable thermocy-cler (PT-100; MJ Research, Inc., Waltham, Massachussets,USA). Reaction mixes were prepared as previouslydescribed [5], with Taq DNA Polymerase, Recombinant(Invitrogen, Carlsbad, California, USA). PCR conditionswere 40 cycles at 94°C (1 min), 49°C (40 sec) and 72°C(35 sec), in a programmable thermocycler (PT-100; MJResearch, Inc.).
RB1 promoter region and exon 1 analysisForward 5'-GGTTTTTCTCAGGGGACGTT-3' and reverse5'-AACCCAGAATCCTGTCACCA-3' primers were used toamplify the RB1 promoter and exon 1 from genomicDNA. Reaction mixes were prepared containing 100 ngDNA, 0.4 pm of each primer (forward and reverse), 5%DMSO, 0.15 mM of each dNTP, 3 mM MgCl2, 1× PCRbuffer (Promega Corporation, Madison, Wisconsin, USA)and 1 U of Platinum Taq DNA polymerase (Promega Cor-poration) in 50 μl reactions. PCR was performed with 40cycles at 94°C (1 min), 50°C (40 sec) and 72°C (45 sec)in a programmable thermocycler (PT-100; MJ Research,Inc., Waltham, Massachussets, USA).
RNA was extracted from blood cells of the proband iso-lated with Ficoll-Paque PLUS® (GE Healthcare UK, LittleChalfont, UK) using TRIZOL® (Invitrogen Corporation,California, USA) following the specifications of the man-ufacturer and cDNA was synthesized with SuperScriptFirst-Strand Synthesis System for RT-PCR (Invitrogen Cor-poration, California, USA).
A cDNA region, including part of the 5' UTR of RB1, wasamplified using the forward primer 5'-CTCCCCG-GCGCTCCTCCACAGC-3' annealing downstream of tran-scription start sites -176 and -128 [10] and the reverseprimer 5'-AGAACACCACGAAAAAGTAA-3' annealing inexon 6. Reaction mixes contained 100 ng cDNA, 0.4 pmof each primer, 5% DMSO, 0.15 mM of each dNTP, 3 mMMgCl2, 1× PCR buffer (Promega Corporation, Madison,Wisconsin, USA) and 1 U of Platinum Taq DNA polymer-ase (Promega Corporation) in 50 μl reactions. PCR wasperformed with 50 cycles at 94°C (1 min), 55°C (40 sec)and 72°C (1 min) in a programmable thermocycler (PT-100; MJ Research, Inc., Waltham, Massachussets, USA).
Amplified products were electrophoresed in 2% agarosegels with ethidium bromide. Direct sequencing was per-formed with an ABI PRISM 377 automatic DNA sequencer
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(Applied Biosystems, Foster City, California, USA).Sequences were analyzed using Sequencher TM DemoVersion 4.1.4 software (Gene Codes Corporation 2005 –775 Technology Drive, Ann Arbor, Michigan, USA) andcompared to a reference sequence (Genbank accessionL11910.1). Constitutive alterations identified in thisstudy were compared with those published in RB1 genemutation database-Retinoblastoma Genetics website(available at http://www.verandi.de/joomla/index.php).
Diagnosis of zygosityGenotyping was carried out with PowerPlexTM16 System(Promega Corporation), following the recommendationsof the manufacturer, for identifying quadruplet zygosity.This assay comprised multiplex PCR amplifications of 15STR loci, including D13S317 (a microsatellite markerlocated at 13q22-q31). An additional microsatellite flan-kling RB1 (D13S284 located at 13q14.3) was also used forgenotyping.
ResultsFundoscopic analysis identified a bilateral retinoblastomain one child (A) while the three other quadruplets andtheir father showed a normal fundoscopy. Mutationalscreening in the proband detected three constitutive alter-ations, one consisting of a transversion in intron 25
(g.174351T>A), previously reported [11,12], and a transi-tion in intron 3 (g.39606 C>T), previously described [7].Both mutations were also present in quadruplets C and Dbut were absent in B and their father (Figure 1). A thirdmutation consisted of a novel transversion at the 5' UTRof the RB1 promoter, in position – 4 (g.2056C>G) withrespect to the initial ATG codon. This mutation was notdetected in her father or the three other quadruplets. Anal-ysis of the proband's cDNA showed a clear electrophero-gram showing only the g.2056C allele (Figure 1).
Genotyping showed that the quadruplets had originatedfrom three trizygotic embryos, one corresponding to A, asecond one to B, and a third one to the monozygotic twinsC and D. One 13q marker (D13S317) included in thePowerPlex 16 system showed two paternal alleles (11 pband 12 pb), and one maternal allele (9 pb) that waspresent in all quadruplets (Table 1). These results wereconfirmed by D13S284, with two paternal alleles (213 pband 215 pb) and two maternal alleles (205 pb and 213pb; see Table 2).
DiscussionSeveral clinical reports have suggested an associationbetween assisted reproduction technologies (ART) andincreased risk of genetic disorders including retinoblast-
1. Partial sequence of 5' UTR of RB1 in genomic DNA showing a C>G mutation (indicated by arrow) in position -4 with respect to the initial translation ATG codon in proband (A)Figure 11. Partial sequence of 5' UTR of RB1 in genomic DNA showing a C>G mutation (indicated by arrow) in posi-tion -4 with respect to the initial translation ATG codon in proband (A). This alteration was not detected in the father or in other quadruplets (B, C and D). 2. Partial sequence of intron 3 in genomic DNA showing a C>T transition (indi-cated by arrow) in proband A and in two monozygotic sisters (C and D). This transition was absent in the father and B. 3. Par-tial sequence of intron 25 in genomic DNA showing a T>A transversion (indicated by arrow) in the proband A and two monozygotic sisters C and D. This transversion was absent in the father and quadruplet B. 4. Partial sequence of 5' UTR of RB1 in cDNA of proband's leukocytes showing only the g.2056C allele.
2 3
FATHER
A
B
C
D
1
A
4
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oma, in children conceived with the use of ART. Thesemight be a consequence of accumulation of epigeneticalterations during embryo culture and/or alteration ofdevelopmental timing. In some cases of retinoblastoma,RB1 was epigenetically silenced by hypermethylation atthe promoter region, resulting in reduced RB1 expression.Since tumor material was not available, peripheral assess-ment of methylation of the RB1 promoter in leukocyteswould not have been informative because the dynamics ofDNA methylation in primary tumor tissues are clearly dif-ferent from normal cells, and not confined to specific CpGsites [13].
RB1 patterns and haplotypesGenotypic analysis of 13q was carried out with three intra-genic RB1 alleles and two microsatellites closely linked toRB1. Presumably, transmission of intragenic allelesoccurred without recombination, corresponding to paren-
tal RB1 patterns [C-C-T] and [C-T-A]. Conversely, the pos-sibility of recombination might have occurred betweenRB1, D13S284 and D13S317.
The putative paternal haplotypes were [C-C-T]-213-11and [C-C-T]-215-12, and the likely maternal haplotypeswere [C-T-A]-213-9 and [C-C-T]-205-9. The mother couldbe a 9/9 homozygous for D13S317 or, alternatively, [C-C-T]-205-9 was a recombinant haplotype, in view that 213-9 cosegregation was more frequent than 205-9.
Parental origin of mutationsThe father was found to be a C/C homozygote at g.2056,while genotypic data indicated that the mother was also aC/C homozygote. Thus, g.2056C>G was not constitu-tional in the proband's progenitors and could haveoccurred either in the maternal or the paternal RB1 allele,as a de novo event in germ lines or in the zygote.
Table 1: Genotyping of four quadruplets and father.
Locus Father Proband (A) Sister (B) Sister (C) Sister (D) Deduced maternal alleles
D3S1358 14–18 17–18 14–17 14–17 14–17 17–?
TH01 7–8 7–9 7–9.3 8–9.3 8–9.3 9–9.3
D21S11 28–29 28–31 29–31 28–31 28–31 31 – ?
D18S51 14–15 14–15 14–15 14–15 14–15 14? – or 15?
PENTA E 13–15 13–15 5–13 13–15 13–15 5 – ?
D5S818 12–12 12–13 12–12 12–13 12–13 12–13
D13S317 11–12 9–11 9–12 9–12 9–12 9 – ?
D7S820 11–12 11–12 11–11 11–12 11–12 11–?
D16S539 8–9 8–12 9–12 9–9 9–9 9–12
CSF1PO 10–12 10–11 10–12 10–10 10–10 11–10
PENTA D 2.2–10 10–13 10–13 2.2–13 2.2–13 13 – ?
VWA 16–16 16–16 13–16 13–16 13–16 13–16
D8S1179 11–13 11–13 9–11 13–13 13–13 9–13
TPOX 8–11 8–11 11–11 8–11 8–11 11 – ?
FGA 21–25 21–21 21–21 21–21 21–21 21–?
AMEL X-Y X-X X-X X-X X-X
? = unknown alleleAnalysis of 16 molecular markers used for genotyping quadruplets and their father. Numbers represent alleles identified with PowerPlex 16 System®. Maternal alleles were deduced. The Amelogenin marker (AMEL) was used as an X chromosome marker. Genotyping allowed for the diagnosis of zygosity in the quadruplets.
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Alternatively, haplotypic data indicated that g.2056C>Gcould have occurred in the maternal RB1 allele becausethe expected paternal haplotype was [C-C-T]-213-11 andthe expected maternal haplotype was [C-T-A]-213-9. Aputative paternal haplotype, identical with the expectedone, could be deduced in the proband, together with aputative maternal [G-T-A]-213-9 haplotype derived fromthe expected maternal haplotype.
Genotypic analysis indicated that both g.39606T andg.174351A alleles were of maternal origin because theywere not present in the father. They were present in threequadruplets derived from two different embryos (A, C andD). Moreover, in the proband, in whom the paternal hap-lotype [C-C-T]-213-11 was deduced, these two mutationsmust have co-segregated with the maternal 213-9 allelesas was the case of quadruplets C and D. Moreover, the factthat child B did not carry these alleles ruled out the possi-bility of co-segregation with the paternal alleles 215-12 inB, C and D (Table 2).
Association of mutations with retinoblastomaWe identified a novel constitutive mutation in the 5' UTRof the RB1 promoter region (g.2056C>G), in position – 4with respect to the initial translation ATG codon in aninfant, born after IVF, and affected with bilateral retino-blastoma. Mutations that weaken adherence to the Kozakconsensus sequence in the 5'UTR have been found to neg-atively affect translation initiation. In eukaryotes,sequences flanking the AUG codon modulate their abilityto halt scanning of the 40S ribosomal subunit and to ini-tiate translation [14]. Translational efficiency was notherein analyzed, but Cs in these positions are highly con-served, being part of the consensus reference. This wasconfirmed by extensive analysis of Genbank data from5'UTR sequences of some 700 vertebrate mRNAs [15].Tumor samples were not available for analysis, a reason
why studies of RB1 expression were not carried out withthis material. cDNA analysis of blood RNA of theproband, however, indicated that only the g.2056C allele,in position -4 and located in the 5' UTR of RB1, wasexpressed, with an apparent null expression of theg.2056G allele.
Mutations detected in intron 3 (g.39606C>T) and intron25 (g.174351T>A) in the proband and her two sisters (Cand D) are less likely to be associated to retinoblastomathan g.2056C>G. The g.39606C>T mutation has beenconsidered to be polymorphic [7] while g.174351T>A wasalso present as a polymorphic trait in unaffected individ-uals, with a frequency of 0.70 for the g.174351T allele and0.30 for the g.174351A allele [12]. Conversely,g.174351T>A was associated to bilateral retinoblastomain one patient in whom this mutation was constitutionaland the only alteration to which retinoblastoma could beassociated [11]. Interestingly, we also found this mutationas the only alteration in seven patients with retinoblast-oma (Barbosa et al., unpublished data), a finding suggest-ing that g.174351T>A might be associated todevelopment of retinoblastoma. Furthermore, the factthat g.174351T>A was also found in the unaffected quad-ruplets C and D does not exclude the possibility of retin-oblastoma at older age.
Genetic counseling and in vitro fertilizationIn this study, retinoblastoma was detected in the probandat one month of age, a reason why the possibility of tumordevelopment could not be excluded in her sisters. This isespecially relevant for the monozygotic infants C and Dwho carried g.39606C>T and g.174351T>A and who mustbe followed by serial fundoscopic analyses. These muta-tions might be associated to retinoblastoma in view ofprevious reports and our unpublished observations.
Table 2: Genotyping of quadruplets and father with respect to mutations and 13q markers.
Mutation/marker Alleles
Father A (proband) B C D
g.2056C>G (in – 4 position in 5'UTR) C/C G/C C/C C/C C/C
g.39606C>T (in intron 3) C/C T/C C/C T/C T/C
g.174351T>A (in intron 25) T/T A/T T/T A/T A/T
D13S284 213/215 213/213 205/215 213/215 213/215
D13S317 11/12 9/11 9/12 9/12 9/12
D13S284 showed two paternal alleles (213 pb and 215 pb) and two maternal alleles (205 pb and 213 pb). The maternal allele 213 pb is present with g.2056G in the proband (A), with g.39606T and with g.174351A in quadruplets A, C and D. Paternal alleles in the quadruplets are indicated in bold characters.
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With respect to IVF, the direct physical manipulation ofgametes and embryos in the ex vivo environment mightresult in the appearance de novo point mutations resultingin the majority of genetic diseases in humans. Moreover,in a study of mouse embryos showed that genetic altera-tions in culture conditions might have considerableimpact on the quality of embryos derived by ART [16].
ConclusionThese results have implications for genetic counseling.Recurrent transmissions or potential transmissions ofMendelian disorders through the same gamete donorhave been reported in ART [17]-[18] and constitutive RB1mutations may appear de novo or be transmitted by oneprogenitor [4]. For establishing an accurate moleculardiagnosis of retinoblastoma, genotypic data are necessary,mainly for distinguishing between hereditary from spo-radic retinoblastoma. In this case, in which blood samplesfrom the oocyte donor was not available, hereditary retin-oblastoma could not be proven.
In ART, occurrence or risk of occurrence of a genetic disor-der in the family must be calculated. Medical, family andreproductive histories of female and male donors must beobtained, and appropriate genetic counseling should beoffered and genetic testing implemented [4]. Theserequirements cannot be met if the anonymity of gametedonors were to be respected with all ethical, legal, andpsychological implications, thus making impracticable aprecise diagnosis for genetic counseling. Our findingrequires further research to confirm the associationbetween IVF and retinoblastoma and to explore likelycausal mechanisms.
Competing interestsThere are no competing interests (political, personal, reli-gious, commercial, ideologic, academic or intellectual orany other) that may influence the authors' findings, opin-ions or points of view.
Authors' contributionsRHB provided sequence and genotypic data of the indi-viduals herein studied and analysed these findings. EL car-ried out fundoscopies and referred the patient and herfamily to FRV for Genetic Counseling. CRB acted as aca-demic supervisor of RHB and contributed to the planningof this study. HNS was involved in drafting and revisingthe manuscript as well as giving final approval to the ver-sion to be published.
AcknowledgementsWe are grateful to Rodrigo Soares de Moura Neto for helping us with gen-otyping and to Anna Claudia Evangelista dos Santos for helping us with blood collections. Work supported by SWISSBRIDGE FOUNDATION (Switzerland), FAPERJ grant E/26 110.944/2008 and CNPq grant 400811/
05-7. RH Barbosa is a recipient of a PhD scholarship from CAPES (Brazilian Coordination for the Improvement of Higher Education Personnel).
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